U.S. patent number 11,150,455 [Application Number 16/621,122] was granted by the patent office on 2021-10-19 for reduced dimensionality structured illumination microscopy with patterned arrays of nanowells.
This patent grant is currently assigned to ILLUMINA CAMBRIDGE LIMITED, ILLUMINA, INC.. The grantee listed for this patent is Illumina Cambridge Limited, Illumina, Inc.. Invention is credited to Geraint Wyn Evans, Stanley S. Hong, Gary Mark Skinner.
United States Patent |
11,150,455 |
Skinner , et al. |
October 19, 2021 |
Reduced dimensionality structured illumination microscopy with
patterned arrays of nanowells
Abstract
Techniques are described for reducing the number of angles
needed in structured illumination imaging of biological samples
through the use of patterned flowcells, where nanowells of the
patterned flowcells are arranged in, e.g., a square array, or an
asymmetrical array. Accordingly, the number of images needed to
resolve details of the biological samples is reduced. Techniques
are also described for combining structured illumination imaging
with line scanning using the patterned flowcells.
Inventors: |
Skinner; Gary Mark (Kedington,
GB), Evans; Geraint Wyn (Cambridge, GB),
Hong; Stanley S. (Palo Alto, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina, Inc.
Illumina Cambridge Limited |
San Diego
Cambridge |
CA
N/A |
US
GB |
|
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Assignee: |
ILLUMINA, INC. (San Diego,
CA)
ILLUMINA CAMBRIDGE LIMITED (Cambridge, GB)
|
Family
ID: |
67395706 |
Appl.
No.: |
16/621,122 |
Filed: |
January 22, 2019 |
PCT
Filed: |
January 22, 2019 |
PCT No.: |
PCT/US2019/014574 |
371(c)(1),(2),(4) Date: |
December 10, 2019 |
PCT
Pub. No.: |
WO2019/147581 |
PCT
Pub. Date: |
August 01, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200103639 A1 |
Apr 2, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62621564 |
Jan 24, 2018 |
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Foreign Application Priority Data
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Mar 20, 2018 [NL] |
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N2020622 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L
3/5085 (20130101); G02B 21/06 (20130101); G02B
27/425 (20130101); B01L 3/50851 (20130101); G01N
21/6452 (20130101); G02B 27/58 (20130101); G01N
21/05 (20130101); B01L 3/50853 (20130101); G01N
21/6458 (20130101); B01L 3/502 (20130101); G02B
21/16 (20130101); G02B 21/365 (20130101); G06V
10/10 (20220101); B01L 2300/0654 (20130101); B01L
2300/0663 (20130101); G01N 2021/6421 (20130101); G01N
2021/6419 (20130101); B01L 2300/0893 (20130101); B01L
2300/0896 (20130101); G01N 2021/6482 (20130101); G01N
21/6408 (20130101) |
Current International
Class: |
G02B
21/06 (20060101); B01L 3/00 (20060101); G02B
27/42 (20060101); G02B 21/36 (20060101); G02B
21/16 (20060101); G01N 21/05 (20060101) |
References Cited
[Referenced By]
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WO |
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Other References
Frohn, J. , et al., "Three-dimensional resolution enhancement in
fluorescence microscopy by harmonic excitation", Optics Letters 26
(11), 2001, 828-830. cited by applicant .
Frohn, J. , et al., "True optical resolution beyond the Rayleigh
limit achieved by standing wave illumination", PNAS 97 (13), 2000,
7232-7236. cited by applicant .
Krishnamurthi, V. , et al., "Image processing in 3D standing-wave
fluorescence microscopy", Three-Dimensional Microscopy: Image
Acquisition and Processing III vol. 2655, International Society for
Optics and Photonics, Apr. 10, 1996, 18-25. cited by
applicant.
|
Primary Examiner: Stafira; Michael P
Attorney, Agent or Firm: Marshall, Gerstein & Borun
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 National Stage of International
Patent Application No. PCT/US2019/014574, filed Jan. 22, 2019,
which itself claims the benefit of and priority to U.S. Provisional
Patent Application No. 62/621,564, filed Jan. 24, 2018, and Dutch
Patent Application No. N2020622, filed Mar. 20, 2018, the content
of each of which is incorporated by reference herein in their
entireties and for all purposes.
Claims
What is claimed is:
1. A method of imaging a biological sample, comprising: projecting
an optical pattern onto a biological sample and capturing a first
image of the optical pattern overlaid on the biological sample, the
biological sample being contained in an asymmetrically patterned
flowcell comprising a plurality of elongated nanowells, wherein
each of the elongated nanowells is elliptically shaped or
rectangularly shaped; phase shifting the projected optical pattern
relative to the biological sample and capturing at least a second
image of the phase shifted optical pattern overlaid on the
biological sample; and reconstructing a high resolution image
representative of the biological sample based upon the first
captured image and the at least second captured image.
2. The method of claim 1, wherein each of the plurality of
elongated nanowells are oriented such that along a first axis of
the asymmetrically patterned flowcell, resolution is increased to
resolve information representative of the biological sample.
3. The method of claim 2, wherein each of the plurality of
elongated nanowells are oriented such that along a second axis of
the asymmetrically patterned flowcell, resolution is not increased
to resolve information representative of the biological sample.
4. The method of claim 1, wherein the capturing of the first and
the at least second images comprises performing line scanning
imaging.
5. The method of claim 4, further comprising: directing light
through an optical diffraction grating in a first phase and angle
orientation, wherein the optical pattern projected onto the
biological sample is an optical diffraction grating pattern
generated by the light being directed through the optical
diffraction grating, wherein phase shifting the projected optical
pattern relative to the biological sample comprises phase shifting
the optical diffraction grating.
6. The method of claim 5, wherein the phase shifting of the optical
diffraction grating comprises phase shifting the optical
diffraction grating along the first angle orientation.
7. The method of claim 6, wherein the phase shifting of the optical
diffraction grating occurs perpendicularly to a direction of the
line scanning imaging.
8. The method of claim 1, further comprising: directing light
through an optical diffraction grating in a first phase and angle
orientation, wherein the optical pattern projected onto the
biological sample is an optical diffraction grating pattern
generated by the light being directed through the optical
diffraction grating, wherein phase shifting the projected optical
pattern relative to the biological sample comprises phase shifting
the optical diffraction grating; and performing a third phase shift
of the optical diffraction grating, projecting the optical
diffraction grating pattern onto the biological sample and
capturing at least a third image of the phase shifted optical
diffraction grating pattern overlaid on the biological sample prior
to reconstructing the high resolution image.
9. A method of imaging a biological sample, comprising: directing
light through an optical diffraction grating in a first phase and
angle orientation; projecting an optical diffraction grating
pattern generated by the light being directed through the optical
diffraction grating onto the biological sample and capturing a
first image of the optical diffraction grating pattern overlaid on
the biological sample, the biological sample being contained in an
asymmetrically patterned flowcell comprising a plurality of
elongated nanowells, wherein each of the elongated nanowells is
elliptically shaped or rectangularly shaped; phase shifting the
optical diffraction grating, projecting the optical diffraction
grating pattern onto the biological sample and capturing at least a
second image of the phase shifted optical diffraction grating
pattern overlaid on the biological sample; reorienting the optical
diffraction grating to a second angle orientation, projecting the
optical diffraction grating pattern onto the biological sample, and
capturing a third image of the optical diffraction grating pattern
overlaid on the biological sample; phase shifting the optical
diffraction grating, projecting the optical diffraction grating
pattern onto the biological sample and capturing at least a fourth
image of the phase shifted optical diffraction grating pattern
overlaid on the biological sample; and reconstructing a high
resolution image representative of the biological sample based upon
the first, the at least second, the third, and the at least fourth
captured images.
10. A system, comprising: a laser source emitting a light beam; an
optical diffraction grating adapted to generate an optical
diffraction grating pattern upon passage of the emitted light beam
through the optical diffraction grating; a camera assembly adapted
to capture a plurality of images of optical diffraction grating
pattern overlaid on a biological sample, the plurality of images
reflecting three phases of the optical diffraction grating relative
to the biological sample, wherein the biological sample is located
in a flowcell comprising a plurality of nanowells that are oriented
in an asymmetrical array and wherein each of the plurality of
nanowells are elliptically shaped or rectangularly shaped; and a
processor adapted to reconstruct a high resolution image
representative of the biological sample based on a combination of
the plurality of images.
11. The system of claim 10, wherein each of the plurality of
nanowells are oriented such that along a first axis of the
flowcell, resolution is increased to resolve information
representative of the biological sample.
12. The system of claim 11, wherein each of the plurality of
nanowells are oriented such that along a second axis of the
flowcell, resolution is not increased to resolve information
representative of the biological sample.
13. A system, comprising: a laser source emitting a light beam; an
optical diffraction grating adapted to generate an optical
diffraction grating pattern upon passage of the emitted light beam
through the optical diffraction grating; a camera assembly adapted
to capture a plurality of images of optical diffraction grating
pattern overlaid on a biological sample, the plurality of images
reflecting three phases of the optical diffraction grating relative
to the biological sample, wherein the camera assembly comprises a
time delay integration line scanning camera assembly; and a
processor adapted to reconstruct a high resolution image
representative of the biological sample based on a combination of
the plurality of images.
14. The system of claim 13, wherein the biological sample is
contained in a flowcell, different portions of which are overlaid
with representations of the three phases of the optical diffraction
grating simultaneously.
15. The system of claim 13, wherein the optical diffraction grating
comprises three phase stepped elements, wherein each of the three
phase stepped elements is adapted to generate an optical
diffraction grating pattern upon passage of the emitted light beam
through the phase stepped element, wherein the camera assembly is
adapted to capture an image of an optical diffraction grating
pattern generated by each of the three phase stepped elements
overlaid on the biological sample.
16. The system of claim 15, wherein the camera assembly comprises
three image sensors, each of the three image sensors adapted to
capture the image of the optical diffraction grating pattern
generated by a respective one of the phase stepped elements.
17. A system, comprising: a laser source emitting a light beam; an
optical diffraction grating adapted to generate an optical
diffraction grating pattern upon passage of the emitted light beam
through the optical diffraction grating; a camera assembly adapted
to capture a plurality of images of optical diffraction grating
pattern overlaid on a biological sample, the plurality of images
reflecting three phases of the optical diffraction grating relative
to the biological sample and two angular orientations of the
optical diffraction grating relative to the biological sample,
wherein the biological sample is located in a flowcell comprising a
plurality of nanowells that are oriented in an asymmetrical array
and wherein each of the plurality of nanowells are elliptically
shaped or rectangularly shaped; and a processor adapted to
reconstruct a high resolution image representative of the
biological sample based on a combination of the plurality of
images.
18. The system of claim 17, wherein each of the plurality of
nanowells are oriented such that resolution is increased to resolve
information representative of the biological sample along first and
second axes of the flowcell.
Description
BACKGROUND
Numerous recent advances in the study of biology have benefited
from improved methods for the analyzing and sequencing of nucleic
acids. For example, the Human Genome Project has determined the
entire sequence of the human genome which, it is hoped, will lead
to further discoveries in fields ranging from treatment of disease
to advances in basic science. A number of new DNA sequencing
technologies have recently been reported that are based on the
massively parallel analysis of unamplified, or amplified single
molecules, either in the form of planar arrays or on beads.
The methodology used to analyze the sequence of the nucleic acids
in such new sequencing techniques is often based on the detection
of fluorescent nucleotides or oligonucleotides. Structured
illumination microscopy (SIM) describes one such sequencing
technique by which spatially structured (i.e., patterned) light may
be used to image a sample in order to increase the lateral
resolution of the microscope by a factor of two or more. During
imaging of the sample, images of the sample may be acquired at
various pattern phases (e.g., at 0.degree., 120.degree., and
240.degree.), with the procedure being repeated by rotating the
pattern orientation about the optical axis (e.g., by 60.degree. and
120.degree.). The captured images (e.g., nine images, one image for
each orientation angle at each pattern phase) may be assembled into
a single image having an extended spatial frequency bandwidth. The
single image may be retransformed into real space to generate an
image having a higher resolution than may normally be resolvable by
the microscope.
In typical implementations of SIM systems, a linearly polarized
light beam is directed through an optical diffraction grating that
diffracts the beam into two or more separate orders that may be
projected on the imaged sample as a sinusoidal interference fringe
pattern. In these implementations, the orientation of the projected
optical diffraction grating pattern is controlled by rotating the
optical diffraction grating about the optical axis, while the phase
of the pattern is adjusted by moving the optical diffraction
grating laterally across the axis. In such systems, the optical
diffraction grating is mounted on a translation stage, which in
turn is mounted on a rotation stage. Additionally, such systems use
a linear polarizer to polarize the light emitted by the light
source before it is received at the grating.
FIG. 1A illustrates an example of a sample 100 and an optical
diffraction grating pattern 102 projected onto sample 100. Although
sample 100 may comprise unresolvable, higher spatial frequencies,
overlaying optical diffraction grating pattern 102 that has a
known, lower spatial frequency on sample 100 results in Moire
fringes. This effectively moves the unresolvable, higher spatial
frequencies to lower spatial frequencies that are resolvable by a
microscope. As described above, capturing images of sample 100 with
different orientations/angles and phases of the optical diffraction
grating pattern 102 relative to sample 100, results in images that
can be combined into a single image that is retransformed into real
space to generate an image having a higher resolution.
SUMMARY
Examples of systems and methods disclosed herein are directed to
techniques for reducing the number of images and dimensions needed
to resolve fluorescent samples using SIM through particularly
patterned flowcells, and the leveraging of light beam movement
relative to the fluorescent samples to achieve an implementation of
SIM that can be used with line scanning techniques.
In accordance with one implementation, a method of imaging a
biological sample, comprises projecting an optical pattern onto a
biological sample and capturing a first image of the optical
pattern overlaid on the biological sample. Additionally, the method
may comprise phase shifting the projected optical pattern relative
to the biological sample, and capturing at least a second image of
the phase shifted optical pattern overlaid on the biological
sample. Further still, the method may comprise reconstructing a
high resolution image representative of the biological sample based
upon the first captured image and the at least second captured
image.
In some examples, the biological sample is contained in an
asymmetrically patterned flowcell comprising a plurality of
elongated nanowells. In some examples, each of the plurality of
elongated nanowells are elliptically shaped or rectangularly
shaped. In some examples, each of the plurality of elongated
nanowells are oriented such that along a first axis of the
asymmetrically patterned flowcell, resolution is increased to
resolve information representative of the biological sample. In
some examples, each of the plurality of elongated nanowells are
oriented such that along a second axis of the asymmetrically
patterned flowcell, resolution is not increased to resolve
information representative of the biological sample.
In some implementations, the capturing of the first and the at
least second images comprises performing line scanning imaging. The
method may further include: directing light through an optical
diffraction grating in a first phase and angle orientation, where
the optical pattern projected onto the biological sample is an
optical diffraction grating pattern generated by the light being
directed through the optical diffraction grating, wherein phase
shifting the projected optical pattern relative to the biological
sample includes phase shifting the optical diffraction grating. The
phase shifting of the optical diffraction grating may comprise
phase shifting the optical diffraction grating along the first
angle orientation. The phase shifting of the optical diffraction
grating can occur perpendicularly to a direction of the line
scanning imaging.
In some examples, the method may further comprise performing a
third phase shift of the optical diffraction grating, projecting
the optical diffraction grating pattern onto the biological sample
and capturing at least a third image of the phase shifted optical
diffraction grating pattern overlaid on the biological sample prior
to reconstructing the high resolution image.
In some examples, a method of imaging a biological sample comprises
directing light through an optical diffraction grating in a first
phase and angle orientation, and projecting an optical diffraction
grating pattern generated by the light being directed through the
optical diffraction grating onto the biological sample and
capturing a first image of the optical diffraction grating pattern
overlaid on the biological sample. The method may further comprise
phase shifting the optical diffraction grating, projecting the
optical diffraction grating pattern onto the biological sample and
capturing at least a second image of the phase shifted optical
diffraction grating pattern overlaid on the biological sample.
Additionally still, the method may comprise reorienting the optical
diffraction grating to a second angle orientation, projecting the
optical diffraction grating pattern onto the biological sample, and
capturing a third image of the optical diffraction grating pattern
overlaid on the biological sample. Moreover, the method may
comprise phase shifting the optical diffraction grating, projecting
the optical diffraction grating pattern onto the biological sample
and capturing at least a fourth image of the phase shifted optical
diffraction grating pattern overlaid on the biological sample.
Furthermore, the method may comprise reconstructing a high
resolution image representative of the biological sample based upon
the first, the at least second, the third, and the at least fourth
captured images.
In some examples, the biological sample is contained in a square
array patterned flowcell comprising a plurality of nanowells.
In some examples, a system may comprise a laser source emitting a
light beam, an optical diffraction grating adapted to generate an
optical diffraction grating pattern upon passage of the emitted
light beam through the optical diffraction grating, and a camera
assembly. The camera assembly can be adapted to capture a plurality
of images of optical diffracting grating pattern overlaid on a
biological sample, the plurality of images reflecting three phases
of the optical diffracting grating relative to the biological
sample. The system may further include a processor adapted to
reconstruct a high resolution image representative of the
biological sample based a combination of the plurality of
images.
In some examples, the biological sample is located in a flowcell
comprising a plurality of nanowells oriented in an asymmetrical
array. In some examples, each of the plurality of nanowells are
elliptically shaped or rectangularly shaped. In some examples, each
of the plurality of nanowells are oriented such that along a first
axis of the flowcell, resolution is increased to resolve
information representative of the biological sample. In some
examples, each of the plurality of nanowells are oriented such that
along a second axis of the flowcell, resolution is not increased to
resolve information representative of the biological sample.
In some examples, the camera assembly comprises a time delay
integration line scanning camera assembly. In some examples, the
biological sample is contained in a flowcell, different portions of
which are overlaid with representations of the three phases of the
optical diffracting grating simultaneously.
In some examples, the optical diffraction grating of the system
includes three phase stepped elements, where each of the three
phase stepped elements is adapted to generate an optical
diffraction grating pattern upon passage of the emitted light beam
through the phase stepped element, where the camera assembly is
adapted to capture an image of an optical diffracting grating
pattern generated by each of the three phase stepped elements
overlaid on the biological sample. In some examples, the camera
assembly includes three image sensors, each of the three image
sensors adapted to capture the image of the optical diffraction
grating pattern generated by a respective one of the phase stepped
elements.
In accordance with another implementation, a system may comprise: a
laser source emitting a light beam; an optical diffraction grating
adapted to generate an optical diffraction grating pattern upon
passage of the emitted light beam through the optical diffraction
grating; and a camera assembly adapted to capture a plurality of
images of optical diffracting grating pattern overlaid on a
biological sample, the plurality of images reflecting three phases
of the optical diffracting grating relative to the biological
sample and two angular orientations of the optical diffraction
grating relative to the biological sample. The system may further
comprise a processor adapted to reconstruct a high resolution image
representative of the biological sample based a combination of the
plurality of images.
In some examples, the biological sample is located in a flowcell
comprising a plurality of nanowells oriented in a square array.
In some examples, each of the plurality of nanowells are oriented
such that along resolution is increased to resolve information
representative of the biological sample along first and second axes
of the flowcell.
It should be appreciated that all combinations of the foregoing
concepts and additional concepts discussed in greater detail below
(provided such concepts are not mutually inconsistent) are
contemplated as being part of the inventive subject matter
disclosed herein. In particular, all combinations of claimed
subject matter appearing at the end of this disclosure are
contemplated as being part of the inventive subject matter
disclosed herein.
Other features and aspects of the disclosed technology will become
apparent from the following detailed description, taken in
conjunction with the accompanying drawings, which illustrate, by
way of example, the features in accordance with implementations of
the disclosed technology. The summary is not intended to limit the
scope of any inventions described herein, which are defined by the
claims and equivalents.
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure, in accordance with one or more various
implementations, is described in detail with reference to the
following figures. The figures are provided for purposes of
illustration only and merely depict typical or example
implementations.
FIG. 1A illustrates one example of structured illumination being
used to lower the frequency pattern of a sample allowing for
increased resolution.
FIG. 1B illustrates, in one example, the number of angles needed to
resolve a sample for imaging.
FIG. 2 illustrates one example of a structured illumination imaging
system.
FIG. 3A illustrates an example of a hexagonal flowcell pattern.
FIG. 3B illustrates an example of a square array flowcell pattern,
the use of which results in reduced dimensionality structured
illumination imaging.
FIG. 3C illustrates an example of an asymmetrical array flowcell
pattern, the use of which results in reduced dimensionality
structured illumination imaging.
FIG. 4 is a flow diagram illustrating example operations that may
be implemented for reduced dimensionality structured illumination
imaging.
FIG. 5 illustrates one example of a line scanning imaging
system.
FIGS. 6A-6C illustrate, in one example, phase shifting of a
structured illumination pattern in one dimension.
FIG. 6D illustrates one example of an asymmetrically pattered
flowcell having different portions simultaneously overlaid with
phase shifted structured illumination patterns
FIG. 7 illustrates an example of a line scanning operation using a
conventionally patterned flowcell.
FIG. 8 illustrates an example of a line scanning imaging system
using a stationary structured illumination pattern.
FIG. 9 illustrates an example of a line scanning operation using a
stationary structured illumination pattern that modulates an
illumination light beam.
FIG. 10 is a flow chart illustrating example operations that may be
implemented for reduced dimensionality structured illumination
imaging used in conjunction with line scanning imaging.
FIG. 11 illustrates an example computing component that may be used
to implement various features of implementations described in the
present disclosure.
FIG. 12 illustrates an example implementation where a grating and
well pattern are configured at a slight angular offset, with three
thin illumination regions projected onto the sample, relatively far
apart.
The figures are not exhaustive and do not limit the present
disclosure to the precise form disclosed.
DETAILED DESCRIPTION
As used herein to refer to diffracted light emitted by a
diffraction grating, the term "order" or "order number" is intended
to mean the number of integer wavelengths that represents the path
length difference of light from adjacent slits of the diffraction
grating for constructive interference. The term "zeroth order" or
"zeroth order maximum" is intended to refer to the central bright
fringe emitted by a diffraction grating in which there is no
diffraction. The term "first-order" is intended to refer to the two
bright fringes emitted on either side of the zeroth order fringe,
where the path length difference is .+-.1 wavelengths.
As used herein to refer to a sample, the term "spot" or "feature"
is intended to mean a point or area in a pattern that can be
distinguished from other points or areas according to relative
location. An individual spot can include one or more molecules of a
particular type. For example, a spot can include a single target
nucleic acid molecule having a particular sequence or a spot can
include several nucleic acid molecules having the same sequence
(and/or complementary sequence, thereof).
As used herein, the term "tile" generally refers to one or more
images of the same region of a sample, where each of the one or
more images represents a respective color channel. A tile may form
an imaging data subset of an imaging data set of one imaging
cycle.
As used herein, the term "x-y plane" is intended to mean a 2
dimensional area defined by straight line axes x and y in a
Cartesian coordinate system. When used in reference to a detector
and an object observed by the detector, the area can be further
specified as being orthogonal to the direction of observation
between the detector and object being detected. When used herein to
refer to a line scanner, the term "y direction" refers to the
direction of scanning.
As used herein, the term "z coordinate" is intended to mean
information that specifies the location of a point, line or area
along an axis that is orthogonal to an x-y plane. In particular
implementations, the z axis is orthogonal to an area of an object
that is observed by a detector. For example, the direction of focus
for an optical system may be specified along the z axis.
As used herein, the term "scan a line" is intended to mean
detecting a 2-dimensional cross-section in an x-y plane of an
object, the cross-section being rectangular or oblong, and causing
relative movement between the cross-section and the object. For
example, in the case of fluorescence imaging an area of an object
having rectangular or oblong shape can be specifically excited (at
the exclusion of other areas) and/or emission from the area can be
specifically acquired (at the exclusion of other areas) at a given
time point in the scan.
Implementations disclosed herein are directed to flowcells
configured to have square or asymmetrical patterns. Recall that SIM
relies on spatially structured (i.e., patterned) light to image a
sample in order to increase the lateral resolution of the
microscope by a factor of two or more. Also recall that
traditionally, images of the sample at multiple pattern phases and
multiple orientations/angles are used to achieve the desired
increase in lateral resolution.
FIG. 1B illustrates generally, in one example, the observable
region of reciprocal space produced by a microscope objective
(which is analogous to its diffraction pattern) and how it is
limited at the edges by the highest spatial frequencies that the
objective can transmit (2NA/.lamda. (graph 120). As illustrated, a
central spot represents the zeroth order component. The zeroth
order and first order diffraction components representing a pattern
of parallel lines are illustrated in graph 122. If the pattern
spacings lie at the limits of resolution, the first order spots
occur at the edge of the observable field (on the k.sub.0
boundary). Due to frequency mixing, the observable regions also
contain, in addition to the normal image of spatial frequencies
(center circle), two new offset frequency images (graph 124) that
are centered on the edge of the original field. These offset images
contain higher spatial frequencies that are not observable using
conventional microscopes. As illustrated by graph 126, a set of
images prepared from three phases at 120.degree. orientations,
ultimately after processing, yield a real image that contains twice
the spatial resolution as may be observed in widefield fluorescence
microscopy.
However, by configuring flowcells to have square or asymmetrical
patterns (rather than hexagonal patterns, for example), fewer
images are needed, as the resolution enhancement required to
resolve the substrate becomes anisotropic, hence constructing an
anisotropic optical transfer function (OTF) through using a more
restricted SIM angle set becomes sufficient to resolve the
substrate to sufficient degree. That is, flowcells having square or
asymmetrical patterns of nanowells allow the axis/axes of a
flowcell having a tighter pitch (i.e., the distance between
immediately adjacent nanowells) and involving increased resolution,
to be aligned with the axis/axes whose resolution is to be
increased. In one example of a square patterned flowcell, increased
resolution is only needed with respect to two axes. Thus, only six
images are needed (an image at each of two angles across three
phases). In the case of an asymmetrically patterned flowcell, only
three images of a sample are needed to achieve increased resolution
(an image at one angle across three phases).
By reducing the number of angles needed to resolve a sample to the
desired degree, the number of images needed to complete imaging of
the sample is reduced. For example, in the context of 4-dye
chemistry, a system may need to acquire 36 images in order to
generate 4 images for base-calling (explained below). The amount of
storage (e.g., disk) space needed to store or cache the captured
images can also be reduced. Additionally still, the processing
and/or computational power needed to assemble the images into a
single image, and then retransform/reconstruct that single image
into one having the desired resolution can also be reduced.
Further still, conventional implementations of SIM are incompatible
with sequencing systems that utilize line scanning techniques to
image a sample. Line scanning can refer to using a line of pixels
that image a flowcell line by line to build a continuous image (as
opposed to a camera or sensor with a two-dimensional array of
pixels that capture a still image of an entire object, e.g., a
flowcell). One particular type of line scanning that lends itself
to sequencing systems is time delay integration (TDI) line
scanning.
With multi-angle SIM implementations, a fixed field of view is
needed to acquire each of the angle/phase image combinations.
However, when images are taken with respect to only a single angle,
as is the case in implementations disclosed herein where an
asymmetrically patterned flowcell is used as a sample substrate,
TDI line scanning can be used to capture images of the sample
covering the three SIM pattern phases. That is, a SIM pattern can
be moved relative to the asymmetrically patterned flowcell to
generate the three phases needed to resolve the sample in the
flowcell with increased resolution along only one axis.
In some implementations, TDI line scanning can be used in
conjunction with SIM techniques to image a sample by using a TDI
line scanning camera or sensor to capture an image along a flowcell
(referred to as a "swath"). That is, TDI line scanning can be
performed on a flowcell patterned with a SIM pattern in a first
phase. The SIM pattern can be shifted to a second phase, and TDI
line scanning can be repeated. The SIM pattern can be shifted to a
third phrase, and TDI line scanning can be repeated again. In this
way, images of the sample at each pattern phase are captured.
Alternatively, different portions of the flowcell can be patterned
with different phases of the SIM pattern. For example, at a first
portion of the flowcell, the SIM pattern can be located in a first
position, at a second portion of the flowcell, the SIM pattern can
be shifted to a second position, and at a third portion of the
flowcell, the SIM pattern can be shifted to a third position. Thus,
as the camera or sensor captures the swath, images of the sample
across each of the three SIM pattern phases are captured in a
single TDI line scan.
Some implementations of TDI line scanning may be implemented with a
three-chip TDI imager where the three phases of a projected fringe
pattern may be specified in one scan. Such implementations may be
implemented using a three-part diffraction grating, where each part
of the diffraction grating corresponds to a specific phase. For
example, a three-element diffraction grating, with each element
phase-stepped, may be formed on the same substrate. By virtue of
this implementation, no movement of the grating or sample may be
needed apart from movement along the scanning direction.
In still other implementations, instead of shifting the SIM pattern
relative to the sample/flowcell, the sample/flowcell is moved while
the SIM pattern remains stationary. It is understood that the
sample is located/placed in the flowcell resulting in the sample
being patterned in accordance with the nanowells making up the
flowcell. When implementing TDI line scanning, as noted above, the
sample/flowcell is already moving. Hence, this movement of the
sample/flowcell can be leveraged to avoid having to shift the SIM
pattern. That is, the movement of the sample/flowcell relative to
the stationary SIM pattern (given the appropriate orientation)
generates the requisite phases needed to resolve the sample.
In some implementations, the grating and well pattern may be
configured at a slight angular offset, with three thin illumination
regions projected onto the sample, relatively far apart. Within
each illumination line, wells may remain predominantly in phase
with the grating, but the distance between the illumination regions
may be sufficient that by the second illumination area they are
lambda/3 out phase, for the phase shift. The spacing between the
illumination lines in such implementations may make it easier to
have 3 image sensors (e.g., three TDI scanner chips) next to each
other. This example scenario is illustrated by FIG. 12.
Before describing various implementations of the systems and
methods disclosed herein in detail, it is useful to describe an
example environment with which the technology disclosed herein can
be implemented. One such example environment is that of a
structured illumination imaging system 200, illustrated in FIG. 2,
that illuminates a sample with spatially structured light. For
example, system 200 may be a structured illumination fluorescence
microscopy system that utilizes spatially structured excitation
light to image a biological sample.
In the example of FIG. 2, a light emitter 250 is configured to
output a light beam that is collimated by collimation lens 251. The
collimated light is structured (patterned) by light structuring
optical assembly 255 and directed by dichroic mirror 260 through
objective lens 242 onto a sample of a sample container 210, which
is positioned on a stage 270. In the case of a fluorescent sample,
the sample fluoresces in response to the structured excitation
light, and the resultant light is collected by objective lens 242
and directed to an image sensor of camera system 240 to detect
fluorescence.
Light structuring optical assembly 255 in various implementations,
further described below, includes one or more optical diffraction
gratings to generate a sinusoidal pattern of diffracted light
(e.g., fringes) that is projected onto samples of a sample
container 210. The diffraction gratings may be one-dimensional or
two-dimensional transmissive, reflective, or phase gratings. As
further described below with reference to particular
implementations, in system 200 the diffraction gratings do not
necessarily involve a rotation stage. In some implementations, the
diffraction gratings may be fixed (e.g., not rotated or moved
linearly) during operation of the imaging system. For example, in a
particular implementation, further described below, the diffraction
gratings may include two fixed one-dimensional transmissive
diffraction gratings oriented substantially or exactly/perfectly
perpendicular to each other (e.g., a horizontal diffraction grating
and vertical diffraction grating).
During each imaging cycle, system 200 utilizes light structuring
optical assembly 255 to acquire a plurality of images at various
phases, displaced laterally along the sample plane (e.g., along x-y
plane), with this procedure repeated one or more times by rotating
the pattern orientation about the optical axis (i.e., with respect
to the x-y plane of the sample). The captured images may then be
spatially reconstructed to generate a higher resolution image
(e.g., an image having about twice the lateral spatial resolution
of individual images).
In system 200, light emitter 250 may be an incoherent light emitter
(e.g., emitting light beams output by one or more excitation
diodes), or a coherent light emitter such as emitter of light
output by one or more lasers or laser diodes. As illustrated in the
example of system 200, light emitter 250 includes an optical fiber
252 for guiding an optical beam to be output. However, other
configurations of a light emitter 250 may be used. In
implementations utilizing structured illumination in a
multi-channel imaging system (e.g., a multi-channel fluorescence
microscope utilizing multiple wavelengths of light), optical fiber
252 may optically couple to a plurality of different light sources
(not shown), each light source emitting light of a different
wavelength. Although system 200 is illustrated as having a single
light emitter 250, in some implementations multiple light emitters
250 may be included. For example, multiple light emitters may be
included in the case of a structured illumination imaging system
that utilizes multiple arms, further discussed below. For example,
light corresponding to different wavelengths, such as blue, green,
red, or other colors may be emitted. In some examples, one light
emitter/source may be used. In some examples, two or more light
emitters/sources may be used.
In some implementations, system 200 may include a tube lens 256
that may include a lens element to articulate along the z-axis to
adjust the structured beam shape and path. For example, a component
of the tube lens may be articulated to account for a range of
sample thicknesses (e.g., different cover glass thickness) of the
sample in container 210.
In the example of system 200, fluid delivery module or device 290
may direct the flow of reagents (e.g., fluorescently labeled
nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and
through) sample container 210 and waste valve 220. Sample container
210 can include one or more substrates upon which the samples are
provided. For example, in the case of a system to analyze a large
number of different nucleic acid sequences, sample container 210
can include one or more substrates on which nucleic acids to be
sequenced are bound, attached or associated. The substrate can
include any inert substrate or matrix to which nucleic acids can be
attached, such as for example glass surfaces, plastic surfaces,
latex, dextran, polystyrene surfaces, polypropylene surfaces,
polyacrylamide gels, gold surfaces, and silicon wafers. In some
applications, the substrate is within a channel or other area at a
plurality of locations formed in a matrix or array across the
sample container 210. System 200 also may include a temperature
station actuator 230 and heater/cooler 235 that can optionally
regulate the temperature of conditions of the fluids within the
sample container 210.
In particular implementations, the sample container 210 may be
implemented as a patterned flowcell including a translucent cover
plate, a substrate, and a liquid contained there between, and a
biological sample may be located at an inside surface of the
translucent cover plate or an inside surface of the substrate. The
flowcell may include a large number (e.g., thousands, millions, or
billions, or more) of wells or regions that are patterned into a
defined array (e.g., a hexagonal array, rectangular array, etc.)
into the substrate. Each region may form a cluster (e.g., a
monoclonal cluster) of a biological sample such as DNA, RNA, or
another genomic material which may be sequenced, for example, using
sequencing by synthesis. The flowcell may be further divided into a
number of spaced apart lanes (e.g., eight lanes), each lane
including a hexagonal array of clusters.
Sample container 210 can be mounted on a sample stage 270 to
provide movement and alignment of the sample container 210 relative
to the objective lens 242. The sample stage can have one or more
actuators to allow it to move in any of three dimensions. For
example, in terms of the Cartesian coordinate system, actuators can
be provided to allow the stage to move in the X, Y and Z directions
relative to the objective lens. This can allow one or more sample
locations on sample container 210 to be positioned in optical
alignment with objective lens 242. Movement of sample stage 270
relative to objective lens 242 can be achieved by moving the sample
stage itself, the objective lens, some other component of the
imaging system, or any combination of the foregoing. Further
implementations may also include moving the entire imaging system
over a stationary sample. Alternatively, sample container 210 may
be fixed during imaging.
In some implementations, a focus (z-axis) component 275 may be
included to control positioning of the optical components relative
to the sample container 210 in the focus direction (typically
referred to as the z axis, or z direction). Focus component 275 can
include one or more actuators physically coupled to the optical
stage or the sample stage, or both, to move sample container 210 on
sample stage 270 relative to the optical components (e.g., the
objective lens 242) to provide proper focusing for the imaging
operation. For example, the actuator may be physically coupled to
the respective stage such as, for example, by mechanical, magnetic,
fluidic or other attachment or contact directly or indirectly to or
with the stage. The one or more actuators can be configured to move
the stage in the z-direction while maintaining the sample stage in
the same plane (e.g., maintaining a level or horizontal attitude,
substantially or perfectly perpendicular to the optical axis). It
can be appreciated that perfect perpendicularity, parallelism, or
other orientation may not be achievable in accordance with some
examples or implementations due to, e.g., manufacturing tolerances,
operational limitations, etc. However, for the purposes of the
technologies disclosed herein, substantially perpendicular,
parallel or other orientation is understood to mean an orientation
sufficient to achieve a desired resolution or other relevant effect
as described and/or contemplated herein. The one or more actuators
can also be configured to tilt the stage. This can be done, for
example, so that sample container 210 can be leveled dynamically to
account for any slope in its surfaces.
The structured light emanating from a test sample at a sample
location being imaged can be directed through dichroic mirror 260
to one or more detectors of camera system 240. In some
implementations, a filter switching assembly 265 with one or more
emission filters may be included, where the one or more emission
filters can be used to pass through particular emission wavelengths
and block (or reflect) other wavelengths. For example, the one or
more emission filters may be used to switch between different
channels of the imaging system. In a particular implementation, the
emission filters may be implemented as dichroic mirrors that direct
emission light of different wavelengths to different image sensors
of camera system 240.
Camera system 240 can include one or more image sensors to monitor
and track the imaging (e.g., sequencing) of sample container 210.
Camera system 240 can be implemented, for example, as a
charge-coupled device (CCD) image sensor camera, but other image
sensor technologies (e.g., active pixel sensor) can be used. Output
data (e.g., images) from camera system 240 may be communicated to a
real time analysis module (not shown) that may be implemented as a
software application that, as further described below, may
reconstruct the images captured during each imaging cycle to create
an image having a higher spatial resolution. As will be described
below, camera system 240 may also be implemented as a TDI CCD
camera to effectuate line scanning techniques.
Although not illustrated, a controller can be provided to control
the operation of structured illumination imaging system 200,
including synchronizing the various optical components of system
200. The controller can be implemented to control aspects of system
operation such as, for example, configuration of light structuring
optical assembly 255 (e.g., selection and/or linear translation of
diffraction gratings), movement of tube lens 256, focusing, stage
movement, and imaging operations. In various implementations, the
controller can be implemented using hardware, algorithms (e.g.,
machine executable instructions), or a combination of the
foregoing. For example, in some implementations the controller can
include one or more CPUs or processors with associated memory. As
another example, the controller can comprise hardware or other
circuitry to control the operation, such as a computer processor
and a non-transitory computer readable medium with machine-readable
instructions stored thereon. For example, this circuitry can
include one or more of the following: field programmable gate array
(FPGA), application specific integrated circuit (ASIC),
programmable logic device (PLD), complex programmable logic device
(CPLD), a programmable logic array (PLA), programmable array logic
(PAL) or other similar processing device or circuitry. As yet
another example, the controller can comprise a combination of this
circuitry with one or more processors.
FIG. 3A illustrates an example configuration of a patterned
flowcell 300 that may be imaged in accordance with implementations
disclosed herein. In this example, flowcell 300 is patterned with a
hexagonal array (see 304) of ordered spots or features 302 that may
be simultaneously imaged during an imaging run. For ease of
illustration, flowcell 300 is illustrated as having tens to
hundreds of spots 302. However, as can be appreciated by one having
skill in the art, flowcell 300 may have thousands, millions, or
billions of spots 302 that are imaged. Moreover, in some instances,
flowcell 300 may be a multi-plane sample comprising multiple planes
(substantially or perfectly perpendicular to focusing direction) of
spots 302 that are sampled during an imaging run. In a particular
implementation, flowcell 300 may be patterned with millions or
billions of wells that are divided into lanes. In this particular
implementation, each well of the flowcell may contain biological
material that is sequenced using sequencing by synthesis.
As alluded to above, in some examples in order to resolve a sample
using patterned flowcell 300, at least nine images are needed to
achieve the requisite resolution. This is because the hexagonal
array of nanowells in patterned flowcell 300 is a high frequency
pattern, where the pitch between nanowells is tight, and
unresolvable. In particular, in this example there are two factors
that can determine how many images are needed to sufficiently
resolve a sample.
The first factor is the number copies of the optical passband that
are desired. Referring back to FIG. 1B, graph 122 shows the normal
passband without the use of SIM. Graph 124 illustrates an example
in which one copy of the optical passband is created. This can
improve resolution in one dimension, while graph 126/graph 306
(FIG. 3A) illustrates an example where three copies of the optical
passband are created, which results in a fairly uniform resolution
improvement in two dimensions.
The second factor is the number of images used to demodulate phases
for each optical passband. Although theoretically, only two images
are needed (to obtain the real and imaginary parts), three images
are typically used to obtain better noise averaging.
It should be understood that when translating an image from spatial
frequency to Fourier space (analysis of raw data generated by a
microscope at the objective rear focal plane is based on Fourier
analysis), the Fourier transform contains 3 components or axes.
That is, the diffraction of light at the objective rear focal plane
creates a diffraction barrier that dictates a maximum resolution of
approximately 200 nm in the lateral (x,y) dimension and 500 nm in
the axial (z) dimension, depending upon the objective numerical
aperture and the average wavelength of illumination. Accordingly,
when using the hexagonal array of nanowells in patterned flowcell
300 images are taken at three angles using SIM. As also discussed
above, in order to obtain the requisite resolution, images must be
taken across three phases at each of the three angles, where the
three phases are needed to ensure all parts on imaging area are
observed (i.e., to cover an entire wavelength of the SIM pattern),
thereby resulting in nine images. This results in increased
resolution in all three axes 308.
However, in one example, using another type of patterned flowcell,
e.g., a flowcell 310, where nanowells 312 are patterned onto a
square array (see 314), only two angles are needed to achieve
increased resolution, the increased resolution being aligned along
the axes of the square array. Graph 316 illustrates an example of
this, where only two copies of the optical passband are created and
needed to achieve the required resolution increase. In other words,
a square patterned flowcell, such as flowcell 310 can be resolved
by aligning the SIM pattern or fringe to those directions in which
an increase in resolution is desired, in this case, along the two
axes (x and y) of the square array. It can be appreciated that
along any diagonal path between neighboring nanowells 312, there
will be some resolution enhancement so that diagonally neighboring
nanowells will be resolvable from one another. However, between
nanowells 312 along the x and y axes, the pitch (P.sub.x, P.sub.y)
is narrow enough that resolution needs to be boosted using SIM,
i.e., the spatial frequency in the x and y axes is too high to be
resolved.
By using a square patterned flowcell, such as flowcell 310, the
dimensionality requirement of conventional sequencing systems using
SIM can be reduced by one dimension, where resolution is increased
in only two axes 318. That is, rather than capture nine images that
cover three angles over three phases each, only six images that
cover two angles over three phases each need to be captured in
order to adequately resolve a sample contained within flowcell 310.
This is advantageous despite a reduction in packing density of
flowcell 310. For example, reduction in packing density may be only
11% over a hexagonal array having the same pitch. However,
implementing SIM in accordance with various examples can result in
a packing density increase of, e.g., 356% for a square patterned
array with a 350 nm pitch, over a non-SIM hexagonal array with a
700 nm pitch.
By using still another type of patterned flowcell, in this example
an asymmetrically patterned flowcell, the dimensionality
requirement of conventional sequencing systems using SIM can be
reduced by yet one more dimension. FIG. 3C illustrates a patterned
flowcell 320 whose nanowells are patterned asymmetrically. In this
implementation, each nanowell 322 is shaped or configured to form
an elongated structure. As utilized herein, the term elongated
structure refers to a shape where the dimension along a first axis
is greater that the dimensions along a second axis. In this
example, the x axis, is narrower than the length or height of
nanowell 322 along another axis (in this example, the y axis). It
should be understood that although the implementation illustrated
in FIG. 3C uses elliptical nanowells, other types of elongated
nanowells, e.g., rectangles, may be used. Any shape of nanowell may
be used that results in a pattern whereby the sample along only one
axis is associated with a resolution increase using SIM. In some
implementations, the dimension of the patterned features that the
fringe width w is at least substantially the same as or slightly
greater than may be a diameter of a circular feature, a length of a
side of a square feature, a length of the longer side or shorter
side of a rectangular feature, a diameter of an elliptical feature
along its major axis or minor axis, or the longest dimension of an
irregularly shaped feature along one axis of the feature (e.g., x
or y axis). In some implementations, the nanowells may
alternatively be shaped as squares or circles, but with asymmetric
spacing therebetween. In various implementations, an asymmetrically
patterned flow cell may refer to an array in which the primary
frequency components are at different distances from the zero
frequency component, an array whose unit cell may be defined by a
variety of pitches, or an array in which the frequency components
of the array may be resolved by an optical transfer function which
is more asymmetric that the traditional 3-angle SIM OTF.
In this way, the sample can be resolved along one direction or
axis, i.e., the y axis, while along another direction or axis,
i.e., the x axis, SIM is used to increase resolution in order to
resolve the sample. That is, along the x axis, the pitch, P.sub.x,
of asymmetrically patterned flowcell 320 is narrow or tight,
entailing an increase in resolution, while along the y axis, the
pitch, P.sub.y, of asymmetrically patterned flow 320 is larger.
Accordingly, resolution is increased in only one direction/along
one axis 318, and only three images are captured in order to
adequately resolve a sample contained within the nanowells of
flowcell 320. Thus, as illustrated by graph 352, only one copy of
the optical passband is created and needed to increase
resolution.
FIG. 4 is a flow chart illustrating example operations that can be
performed in a sequencing system, such as structured illumination
imaging system 200 of FIG. 2, to sequence a sample using a square
or asymmetrically patterned flowcell. At operation 400, a light
source corresponding to a first optical diffraction grating pattern
oriented in a first phase may be turned on. At operation 410, the
optical diffraction grating pattern in a first orientation is
projected onto a sample and an image is captured. That is,
referring back to FIG. 2, light emitter 250 can output a light beam
that is collimated by collimation lens 251. The collimated light is
structured (patterned) by light structuring optical assembly 255
and directed by dichroic mirror 260 through objective lens 242 onto
a sample of sample container 210, which is positioned on a stage
270. In this implementation, sample container 210 comprises a
patterned flowcell having a square or asymmetrical pattern, such as
flowcells 310 or 320, respectively (FIGS. 3B and 3C). In the case
of a fluorescent sample, the sample contained in the square or
asymmetrically patterned flowcell fluoresces in response to the
structured excitation light, and the resultant light is collected
by objective lens 242 and directed to an image sensor of camera
system 240 to detect fluorescence.
At operation 420, a check can be performed to determine if an
additional phase shift is needed. If so, at operation 430, the
optical diffraction grating is phase shifted, and operation returns
to operation 410, where the optical diffraction grating pattern
(phase shifted) is projected onto the sample, and an image is
captured. As described previously, three phase shifts are generally
performed to capture an entire imaging area, in this
implementation, the entire area of the square patterned
flowcell.
If no additional phase shift is needed, at operation 440, a check
can be performed to determine if an additional angle is needed, and
the angle of the optical diffraction grating is changed at
operation 450. Operation returns to operation 410, where the
optical diffraction grating pattern (after changing angles) is
projected onto the sample, and an image is captured. Operation
proceeds to operation 420, where if an additional phase shift is
needed at 420, the optical diffraction grating is phase shifted at
operation 430. Again, operation returns to operation 410, where the
optical diffraction grating pattern (at a new angle and new phase)
is projected onto the sample, and an image is captured. Again, in
this implementation, images over three phases are needed to capture
the entire are of the square patterned flowcell. It should be
understood that the aforementioned controller used to control
aspects of system operation of structured illumination imaging
system 200 can be configured with instructions to perform the
above-described functions, e.g., checking whether or not additional
phase shifts or orientations of the optical diffraction grating
pattern are needed to image the particular type of flowcell being
used.
In the case of a square patterned flowcell, e.g., flowcell 310
(FIG. 3), images at two angles are needed to increase resolution
along the two axes of flowcell 310. Accordingly, after capturing
images with the optical diffraction grating pattern projected in
two orientations corresponding to the two angles (over three phase
shifts of the optical diffraction grating pattern), a high
resolution image is reconstructed at operation 460 (by combining
the six total images and retransforming them into real space. This
high resolution image reconstruction can be done in-system, or in
some examples, reconstruction can be performed using a separate
processing entity.
In an implementation where the patterned flowcell is an
asymmetrical flowcell, the above-described method need not involve
changing angles. Again, with an asymmetrical flowcell, SIM is used
to increase resolution along only one axis. Accordingly, the
optical diffraction grating need only be phase shifted three times,
allowing images to be captured for the three phase shifts.
Accordingly, once no other phase shifts are needed at operation
420, the method proceeds to operation 460, where a high resolution
image can be reconstructed using only the three captured
images.
As previously indicated, when using particularly patterned
flowcells that can take advantage of reduced dimensionality SIM
implementations, line scanning techniques, such as TDI line
scanning, can be used to image samples contained in those patterned
flowcells. FIG. 5 is block diagram illustrating an example
two-channel, line scanning imaging system 500 that may be used to
image a sample in various implementations.
As in the case of structured illumination imaging system 200 of
FIG. 2, line scanning imaging system 500 may be used for the
sequencing of nucleic acids, where those where nucleic acids are
attached at fixed locations in an array (i.e., the wells of a
flowcell, such as flowcell 320) and the array can be imaged
repeatedly. In such implementations, line scanning imaging system
500 may obtain images in two different color channels, which may be
used to distinguish a particular nucleotide base type from another.
More particularly, line scanning imaging system 500 may implement a
process referred to as "base calling," which generally refers to a
process of a determining a base call (e.g., adenine (A), cytosine
(C), guanine (G), or thymine (T)) for a given spot location of an
image at an imaging cycle. During two-channel base calling, image
data extracted from two images may be used to determine the
presence of one of four base types by encoding base identity as a
combination of the intensities of the two images. For a given spot
or location in each of the two images, base identity may be
determined based on whether the combination of signal identities is
[on, on], [on, off], [off, on], or [off, off].
Referring again to line scanning imaging system 500, the system
includes a line generation module LGC 510 with two light sources,
511 and 512, disposed therein. Light sources 511 and 512 may be
coherent light sources such as laser diodes which output laser
beams. Light source 511 may emit light in a first wavelength (e.g.,
a red color wavelength), and light source 512 may emit light in a
second wavelength (e.g., a green color wavelength). The light beams
output from laser sources 511 and 512 may be directed through a
beam shaping lens or lenses 513. In some implementations, a single
light shaping lens may be used to shape the light beams output from
both light sources. In other implementations, a separate beam
shaping lens may be used for each light beam. In some examples, the
beam shaping lens is a Powell lens, such that the light beams are
shaped into line patterns. The beam shaping lenses of LGC 510 or
other optical components imaging system be configured to shape the
light emitted by light sources 511 and 512 into a line patterns
(e.g., by using one or more Powel lenses, or other beam shaping
lenses, diffractive or scattering components). For example, in some
implementations light emitted by light sources 511 and 512 can be
sent through an optical diffraction grating to generate an optical
diffraction grating pattern (SIM pattern) that can be projected
onto a sample.
LGC 510 may further include mirror 514 and semi-reflective mirror
515 configured to direct the light beams through a single interface
port to an emission optics module (EOM) 530. The light beams may
pass through a shutter element 516. EOM 530 may include objective
535 and a z-stage 536 which moves objective 535 longitudinally
closer to or further away from a target 550. For example, target
(e.g., a patterned flowcell) 550 may include a liquid layer 552 and
a translucent cover plate 551, and a biological sample may be
located at an inside surface of the translucent cover plate as well
an inside surface of the substrate layer located below the liquid
layer. The z-stage may then move the objective as to focus the
light beams onto either inside surface of the flowcell (e.g.,
focused on the biological sample). The biological sample may be
DNA, RNA, proteins, or other biological materials responsive to
optical sequencing as known in the art.
EOM 530 may include semi-reflective mirror 533 to reflect a focus
tracking light beam emitted from a focus tracking module (FTM) 540
onto target 550, and then to reflect light returned from target 550
back into FTM 540. FTM 540 may include a focus tracking optical
sensor to detect characteristics of the returned focus tracking
light beam and generate a feedback signal to optimize focus of
objective 535 on target 550.
EOM 530 may also include semi-reflective mirror 534 to direct light
through objective 535, while allowing light returned from target
550 to pass through. In some implementations, EOM 530 may include a
tube lens 532. Light transmitted through tube lens 532 may pass
through filter element 531 and into camera assembly 520. Camera
assembly 520 may include one or more optical sensors 521, e.g., TDI
line scanning sensors, to detect light emitted from the biological
sample in response to the incident light beams (e.g., fluorescence
in response to red and green light received from light sources 511
and 512). In one example, an LGC (such as that described above) may
project light through a diffraction grating to generate a linear
fringe pattern.
Output data from the sensors of camera assembly 520 may be
communicated to a real time analysis circuit 525. Real time
analysis circuit 525, in various implementations, executes computer
readable instructions for analyzing the image data (e.g., image
quality scoring, base calling, etc.), reporting or displaying the
characteristics of the beam (e.g., focus, shape, intensity, power,
brightness, position) to a graphical user interface (GUI), etc.
These operations may be performed in real-time during imaging
cycles to minimize downstream analysis time and provide real time
feedback and troubleshooting during an imaging run. In
implementations, real time analysis circuit 525 may be a computing
device (e.g., computing device 1100) that is communicatively
coupled to and controls imaging system 500. In implementations
further described below, real time analysis circuit 525 may
additionally execute computer readable instructions for correcting
distortion in the output image data received from camera assembly
520.
FIGS. 6A-6C represent an example representation of TDI line
scanning of an asymmetrically patterned flowcell, where SIM is used
to increase resolution along one axis of the flowcell. In
particular, FIG. 6A illustrates an asymmetrically patterned
flowcell 620 (which may be an implementation of asymmetrically
patterned flowcell 320 (FIG. 3C) on which a SIM pattern 630 is
overlaid. TDI line scanning can be performed along the y axis, to
capture row-by-row images of the asymmetrically patterned flowcell
620. The images captured in FIG. 6A are captured with SIM pattern
630 in a first phase.
By way of example, line scanning imaging system 500 may use LGC 510
in coordination with the optics of the system to line scan the
sample (overlaid with a SIM pattern, i.e., an optical diffraction
grating pattern) with light having wavelengths within the red color
spectrum and to line scan the sample with light having wavelengths
within the green color spectrum. In response to line scanning,
fluorescent dyes situated at the different spots of the sample may
fluoresce and the resultant light may be collected by the objective
lens 535 and directed to an image sensor of camera assembly 520 to
detect the florescence. For example, fluorescence of each spot may
be detected by a few pixels of camera assembly 520. Image data
output from camera assembly 520 may then be communicated to real
time analysis circuit 525 for processing, e.g., to combine the
images to form a swath.
FIG. 6B illustrates asymmetrically patterned flowcell 620 overlaid
with SIM pattern 630. However, in FIG. 6B, SIM pattern 630 has been
phase shifted along the x axis (in alignment with the axis needing
a resolution increase to resolve the sample). As described above,
line scanning imaging system 500 may use LGC 510 in coordination
with the optics of the system to line scan the sample (overlaid
with phase shifted SIM pattern 630). Images may be captured and
output from camera assembly 520 and again communicated to real time
analysis circuit 525 for processing.
FIG. 6C illustrates asymmetrically patterned flowcell 620 overlaid
with SIM pattern 630. In FIG. 6C, SIM pattern 630 has been phase
shifted to a third phase along the x axis (in alignment with the
axis needing a resolution increase to resolve the sample). Again,
line scanning imaging system 500 may use LGC 510 in coordination
with the optics of the system to line scan the sample (overlaid
with phase shifted SIM pattern 630). Images may be captured and
output from camera assembly 520 and again communicated to real time
analysis circuit 525 for processing. The images captured in
accordance with each phase/phase shift may be combined by real time
analysis circuit 525 into a single image and retransformed into
real space to generate an image having a higher resolution, in this
example, along the x axis.
In another implementation, as illustrated in FIG. 6D, different
portions of flowcell 620 can be overlaid with SIM pattern 630 in
its different phases. That is, a SIM pattern in a first phase 630A
is overlaid along a lower portion of flowcell 620, the same SIM
pattern in a second phase 630B is overlaid along a middle portion
of flowcell 620, and again, the same SIM pattern in a third phase
630C is overlaid along an upper portion of flowcell 620.
Accordingly line scanning imaging system 500 line scans flowcell
620 overlaid with the different phases of a SIM pattern,
(630A-630B), such that line scanning imaging system 500 can image
the entire flow, in accordance with each requisite phase of the SIM
pattern, in a single run. In some implementations, line scanning
imaging system 500 can be modified to have multiple LGCs and
multiple cameras or sensors/camera assemblies, e.g., three, each of
which generate and output light through three optical diffraction
gratings (the same but oriented in different phases) to generate
the three phases of the SIM pattern. In this way, each camera or
sensor/camera assembly is able to capture an image of flowcell 620
along with a different SIM pattern phase simultaneously.
As alluded to above, in still other implementations, a
sample/flowcell can be moved while the SIM pattern remains
stationary. When implementing TDI line scanning, the
sample/flowcell is already moving. Hence, this movement of the
sample/flowcell can be leveraged to avoid having to shift the SIM
pattern. That is, the movement of the sample/flowcell relative to
the stationary SIM pattern generates the requisite phases needed to
resolve the sample.
FIG. 7 illustrates another example patterned flowcell 720, similar
to the hexagonal array patterned flowcell 300 (FIG. 3A). In a
conventional structured illumination imaging system, flowcell 720
can be line scanned, e.g., in the direction of the y axis.
Intensity of a light beam output by an LGC, e.g., LGC 510 (FIG. 5)
onto the sample in flowcell 720 is shown as being wide and
homogenous along the x axis (not shown, but substantially or
exactly perpendicular to the line scanning direction). Along the y
axis, however, the intensity of the light beam is narrow. As the
laser beam moves relative to flowcell 720, fluorescence images are
captured by a line scanning camera or sensor, e.g., camera assembly
520 (FIG. 5) in the corresponding area being illuminated by the
light beam.
However, taking advantage of the fact that the sample/flowcell 720
is already moving, and because only one dimensional SIM is needed
to resolve samples in an asymmetrically patterned flowcell, e.g.,
flowcell 320 (FIG. 3C), the optical diffraction grating that
produces the SIM pattern can be kept still. That is, the requisite
multiple (e.g., three) phases needed to adequately resolve the
sample. Accordingly, moving stages or other elements needed for
moving, e.g., a rotating or translating the optical diffraction
grating, in a conventional line scanning imaging system are not
needed in this implementation.
FIG. 8 illustrates an example line scanning imaging system 800 that
uses a stationary optical diffraction grating. It should be noted
that, for ease of explanation, FIG. 8 is a simplified illustration
in which not all features/elements are shown. However, line
scanning system 800 may be one implementation of line scanning
imaging system 500 that uses a stationary optical diffraction
grating to keep the resulting optical diffraction grating
pattern/SIM pattern still.
In the example of FIG. 8, a light emitter, e.g., laser 802, is
configured to output a light beam that is collimated by collimation
lens 804. In one implementation, laser 802 emits light in the green
wavelength. The collimated light is directed by dichroic filter 806
through a stationary optical diffraction grating 812 to objective
lens 830 via another dichroic filter 828 onto a sample of a sample
container 832. In this implementation, sample container 830 is an
asymmetrically patterned flow cell, such as flowcell 320 (FIG.
3C).
A second light emitter, e.g., laser 808, emits light (in the red
wavelength, for example) through stationary optical diffraction
grating 812 to objective lens 830, also via dichroic filter 828,
and onto the sample of sample container 832. Sample container 832
is positioned on a stage 840 that can move sample container 832
relative to the light beams from lasers 802 and 808. In the case of
a fluorescent sample, the sample fluoresces in response to the
structured excitation light (laser beams from lasers 802 and 808),
and the resultant light is collected by objective lens 828 and
directed to an image sensor of cameras 814 and 820.
Dichroic filter 806 is used to pass the green light beam from laser
802 to pass on through to stationary optical diffraction grating
812, while reflecting the red light beam from laser 808 towards
stationary optical diffraction grating 812. Dichroic filter 828
functions similarly in that it allows the red and green light beams
from lasers 802 and 808 to be reflected to objective lens 830,
while allowing camera 814 and 820 to respectively capture images
fluoresced with the green and red light. Dichroic filter 816
directs green light emissions from the fluoresced sample to camera
814, while dichroic filter 822 directs red light emissions from the
fluoresced sample to camera 820. Lenses 818 and 824 are collimating
lens for cameras 814 and 820, respectively. Dichroic mirror 826
directs the green and red light emissions from the fluoresced
sample to the appropriate cameras.
In line scanning system 800, optical diffraction grating 812 is
stationary. That is, as previously discussed, by using
asymmetrically patterned flowcells in conjunction with SIM, only
one dimension of structured illumination is needed, and multiple
phases can be achieved by moving the beam along the flowcell. In
other words, movement of the laser beam relative to the
sample/flowcell or movement of the sample/flowcell relative to the
laser beam, resulting in the relative movement between sample and
fringe excitation patterns is all that is needed to generate the
different phases.
FIG. 9 illustrates a patterned flowcell 920 that may be line
scanned with a line scanning imaging system, such as line scanning
system 800. An optical diffraction grating pattern can be projected
onto flowcell 920, while flowcell 920 moves in accordance with line
scanning imaging techniques. Movement of flowcell 920 relative to
the stationary optical diffraction grating pattern creates the
necessary phase shifts and the images captured during line
scanning, once combined and retransformed into real space increase
the resolution, as previously discussed.
In particular, the light beam moves in the direction of the y axis.
Again, intensity of the light beam is homogenous along the x axis
(not shown), but the intensity along the y axis is modulated due to
passage through a stationary optical diffraction grating, e.g.,
stationary optical diffraction grating 812 (FIG. 8). As the light
beam moves relative to flowcell 920, the optical diffraction
grating pattern shifts. In fact, more than three, or even dozens of
phase shifts can be generated. As a result, by moving the
sample/flowcell 920 instead of the optical diffraction grating, an
increase in resolution along the axis of the line scanning can be
achieved. In some implementations, as described above, resolution
in this direction can be increased by at least two times on
surfaces with either both random features or periodic patterns. It
should be understood that because the resolution can be increased,
e.g., by at least two times, the density of the nanowells in
flowcell 920 can be increased by a factor of two or more.
FIG. 10 is a flow chart illustrating example operations that can be
performed in a line scanning imaging system, such as line scanning
imaging system 500 (FIG. 5) or line scanning imaging system 800
(FIG. 8), to sequence a sample using an asymmetrically patterned
flowcell. At operation 1000, light beams from laser sources, e.g.,
laser sources 802 and 808, are output through a stationary optical
diffraction grating, e.g., stationary optical diffraction grating
812, corresponding to a first optical diffraction grating pattern
orientation may be turned on. At operation 1010, the optical
diffraction grating pattern is projected onto a sample, and at
operation 1020, the sample is line scanned. Line scanning may be
performed as previously described with regarding to line scanning
imaging system 800 (FIG. 8). At operation 1030, the sample is moved
in accordance the aforementioned line scanning techniques or the
directed light may be moved as also described above to achieve
relative motion between the sample and optical diffraction grating
pattern.
Operations 1020 and 1030 may be repeated as many times as necessary
to capture images representative of the entire sample. Again, as a
result of the sample being moved relative to the stationary optical
diffraction grating pattern, images of the sample and optical
diffraction grating pattern can be captured across the requisite
phase shifts needed to increase resolution. At operation 1040, a
high resolution image can be reconstructed.
It should be noted that in order to prevent motion blur between the
optical diffraction grating pattern and the sample during line
scanning, the laser sources can operate in a pulsed fashion. That
is, the laser sources, e.g., laser sources 802 and 808 may be
pulsed so that at every excitation, a line scanning image can be
captured. In some implementations, the orientation of the optical
diffraction grating pattern relative to the sample/flowcell can be
shifted by 90.degree.. In other implementations, as illustrated in
FIGS. 6A-6C, if the orientation of the optical diffraction grating
pattern is such that the sample is not moving through areas of
light and dark (as may be the case if the orientation of the
optical diffraction grating pattern was shifted by 90.degree.),
pulsing of the laser sources may not be needed because movement of
the sample relative to the optical diffraction grating pattern
moves through the same fringe intensity.
It should be noted that, although implementations described herein
have been primarily described in the context of using diffraction
gratings to create fringe patterns that are projected onto an
imaged sample, in implementations the projected fringe patterns
need not necessarily be created by diffraction gratings. Any method
of creating a sinusoidal fringe pattern may be suitable. Creation
of a fringe pattern may be achieved via interference between two
counter propagating beams, mutually coherent at the point of the
desired interference pattern; via coherent or incoherent imaging of
a diffraction grating; via beams separated via a beam splitter and
interfered; counter propagating beams in a light-pipe or waveguide,
etc.
FIG. 11 illustrates an example computing component that may be used
to implement various features of the system and methods disclosed
herein, such as the aforementioned features and functionality of
one or more aspects of the methods illustrated in FIGS. 4 and 10
implemented in systems 200, 500, and/or 800 and described herein.
For example, computing component may be implemented as a real-time
analysis circuit 525.
As used herein, the term circuit might describe a given unit of
functionality that can be performed in accordance with one or more
implementations of the present application. As used herein, a
circuit might be implemented utilizing any form of hardware or a
combination of hardware and software. For example, one or more
processors, controllers, ASICs, PLAs, PALs, CPLDs, FPGAs, logical
components, software routines or other mechanisms might be
implemented to make up a circuit. In implementation, the various
circuits described herein might be implemented as discrete circuits
or the functions and features described can be shared in part or in
total among one or more circuits. In other words, one of ordinary
skill in the art after reading this description, can appreciate
that the various features and functionality described herein may be
implemented in any given application and can be implemented in one
or more separate or shared circuits in various combinations and
permutations. Even though various features or elements of
functionality may be individually described or claimed as separate
modules, one of ordinary skill in the art will understand that
these features and functionality can be shared among one or more
common software and hardware elements, and such description shall
not require or imply that separate hardware or software components
are used to implement such features or functionality.
Where components or circuits of the application are implemented in
whole or in part using software, in one implementation, these
software elements can be implemented to operate with a computing or
processing module capable of carrying out the functionality
described with respect thereto. One such example computing
component is shown in FIG. 13. Various implementations are
described in terms of this example-computing component 1000. After
reading this description, it will become apparent to a person
skilled in the relevant art how to implement the application using
other computing modules or architectures.
Referring now to FIG. 13, computing component 1000 may represent,
for example, computing or processing capabilities found within
desktop, laptop, notebook, and tablet computers; hand-held
computing devices (tablets, PDA's, smart phones, cell phones,
palmtops, etc.); mainframes, supercomputers, workstations or
servers; or any other type of special-purpose or general-purpose
computing devices as may be desirable or appropriate for a given
application or environment. Computing component 1000 might also
represent computing capabilities embedded within or otherwise
available to a given device. For example, a computing component
might be found in other electronic devices such as, for example,
digital cameras, navigation systems, cellular telephones, portable
computing devices, modems, routers, WAPs, terminals and other
electronic devices that might include some form of processing
capability.
Computing component 1000 might include, for example, one or more
processors, controllers, control modules, or other processing
devices, such as a processor 1004. Processor 1004 might be
implemented using a general-purpose or special-purpose processing
engine such as, for example, a microprocessor, controller, or other
control logic. In the illustrated example, processor 1004 is
connected to a bus 1002, although any communication medium can be
used to facilitate interaction with other components of computing
component 1000 or to communicate externally.
Computing component 1000 might also include one or more memory
modules, simply referred to herein as main memory 1008. For
example, preferably random access memory (RAM) or other dynamic
memory, might be used for storing information and instructions to
be executed by processor 1004. Main memory 1008 might also be used
for storing temporary variables or other intermediate information
during execution of instructions to be executed by processor 1004.
Computing component 1000 might likewise include a read only memory
("ROM") or other static storage device coupled to bus 1002 for
storing static information and instructions for processor 1004.
The computing component 1000 might also include one or more various
forms of information storage mechanism 1010, which might include,
for example, a media drive 1012 and a storage unit interface 1020.
The media drive 1012 might include a drive or other mechanism to
support fixed or removable storage media 1014. For example, a hard
disk drive, a solid state drive, a magnetic tape drive, an optical
disk drive, a CD or DVD drive (R or RW), or other removable or
fixed media drive might be provided. Accordingly, storage media
1014 might include, for example, a hard disk, a solid state drive,
magnetic tape, cartridge, optical disk, a CD, DVD, or Blu-ray, or
other fixed or removable medium that is read by, written to or
accessed by media drive 1012. As these examples illustrate, the
storage media 1014 can include a computer usable storage medium
having stored therein computer software or data.
In alternative examples, information storage mechanism 1010 might
include other similar instrumentalities for allowing computer
programs or other instructions or data to be loaded into computing
component 1000. Such instrumentalities might include, for example,
a fixed or removable storage unit 1022 and an interface 1020.
Examples of such storage units 1022 and interfaces 1020 can include
a program cartridge and cartridge interface, a removable memory
(for example, a flash memory or other removable memory module) and
memory slot, a PCMCIA slot and card, and other fixed or removable
storage units 1022 and interfaces 1020 that allow software and data
to be transferred from the storage unit 1022 to computing component
1000.
Computing component 1000 might also include a communications
interface 1024. Communications interface 1024 might be used to
allow software and data to be transferred between computing
component 1000 and external devices. Examples of communications
interface 1024 might include a modem or softmodem, a network
interface (such as an Ethernet, network interface card, WiMedia,
IEEE 802.XX or other interface), a communications port (such as for
example, a USB port, IR port, RS232 port Bluetooth.RTM. interface,
or other port), or other communications interface. Software and
data transferred via communications interface 1024 might typically
be carried on signals, which can be electronic, electromagnetic
(which includes optical) or other signals capable of being
exchanged by a given communications interface 1024. These signals
might be provided to communications interface 1024 via a channel
1028. This channel 1028 might carry signals and might be
implemented using a wired or wireless communication medium. Some
examples of a channel might include a phone line, a cellular link,
an RF link, an optical link, a network interface, a local or wide
area network, and other wired or wireless communications
channels.
In this document, the terms "computer readable medium", "computer
usable medium" and "computer program medium" are used to generally
refer to non-transitory media, volatile or non-volatile, such as,
for example, memory 1008, storage unit 1022, and media 1014. These
and other various forms of computer program media or computer
usable media may be involved in carrying one or more sequences of
one or more instructions to a processing device for execution. Such
instructions embodied on the medium, are generally referred to as
"computer program code" or a "computer program product" (which may
be grouped in the form of computer programs or other groupings).
When executed, such instructions might enable the computing module
1000 to perform features or functions of the present application as
discussed herein.
Although described above in terms of various examples and
implementations, it should be understood that the various features,
aspects and functionality described in one or more of the
individual implementations are not limited in their applicability
to the particular implementation with which they are described, but
instead can be applied, alone or in various combinations, to one or
more of the other implementations of the application, whether or
not such implementations are described and whether or not such
features are presented as being a part of a described
implementation. Thus, the breadth and scope of the present
application should not be limited by any of the above-described
example implementations.
It should be appreciated that all combinations of the foregoing
concepts (provided such concepts are not mutually inconsistent) are
contemplated as being part of the inventive subject matter
disclosed herein. In particular, all combinations of claimed
subject matter appearing at the end of this disclosure are
contemplated as being part of the inventive subject matter
disclosed herein.
The terms "substantially" and "about" used throughout this
disclosure, including the claims, are used to describe and account
for small fluctuations, such as due to variations in processing.
For example, they can refer to less than or equal to .+-.5%, such
as less than or equal to .+-.2%, such as less than or equal to
.+-.1%, such as less than or equal to .+-.0.5%, such as less than
or equal to .+-.0.2%, such as less than or equal to .+-.0.1%, such
as less than or equal to .+-.0.05%.
To the extent applicable, the terms "first," "second," "third,"
etc. herein are merely employed to show the respective objects
described by these terms as separate entities and are not meant to
connote a sense of chronological order, unless stated explicitly
otherwise herein.
Terms and phrases used in this document, and variations thereof,
unless otherwise expressly stated, should be construed as open
ended as opposed to limiting. As examples of the foregoing: the
term "including" should be read as meaning "including, without
limitation" or the like; the term "example" is used to provide
example instances of the item in discussion, not an exhaustive or
limiting list thereof; the terms "a" or "an" should be read as
meaning "at least one," "one or more" or the like; and adjectives
such as "conventional," "traditional," "normal," "standard,"
"known" and terms of similar meaning should not be construed as
limiting the item described to a given time period or to an item
available as of a given time, but instead should be read to
encompass conventional, traditional, normal, or standard
technologies that may be available or known now or at any time in
the future. Likewise, where this document refers to technologies
that may be apparent or known to one of ordinary skill in the art,
such technologies encompass those apparent or known to the skilled
artisan now or at any time in the future.
The presence of broadening words and phrases such as "one or more,"
"at least," "but not limited to" or other like phrases in some
instances shall not be read to mean that the narrower case is
intended or required in instances where such broadening phrases may
be absent.
Additionally, the various implementations set forth herein are
described in terms of example block diagrams, flow charts and other
illustrations. As will become apparent to one of ordinary skill in
the art after reading this document, the illustrated
implementations and their various alternatives can be implemented
without confinement to the illustrated examples. For example, block
diagrams and their accompanying description should not be construed
as mandating a particular architecture or configuration.
While various implementations of the present disclosure have been
described above, it should be understood that they have been
presented by way of example only, and not of limitation. Likewise,
the various diagrams may depict an example architectural or other
configuration for the disclosure, which is done to aid in
understanding the features and functionality that can be included
in the disclosure. The disclosure is not restricted to the
illustrated example architectures or configurations, but the
desired features can be implemented using a variety of alternative
architectures and configurations. Indeed, it will be apparent to
one of skill in the art how alternative functional, logical or
physical partitioning and configurations can be implemented to
implement the desired features of the present disclosure. Also, a
multitude of different constituent component names other than those
depicted herein can be applied to the various partitions.
Additionally, with regard to flow diagrams, operational
descriptions and method claims, the order in which the steps are
presented herein shall not mandate that various implementations be
implemented to perform the recited functionality in the same order
unless the context dictates otherwise.
* * * * *